Rationale: My plaque assay turned out negative, so I collected new soil. This time from a living tree rather than a dead tree, but still from Cameron Park and still sick. I also collected metadata on the soil so to find if that may be a confounding variable.

Procedure: Soil Metadata:

1. After collecting data from tree stored in refrigerator overnight.
2. pH tested soil and put a 4.05g sample of soil in a weight boat to let dry for

   48 hours

3. Placed 10mL of soil in falcon tube and filled to 30mL with DI water then

   poured 3 drops of dispersion fluid and let sit under vent hood for 48 hours

4. After 48 hours of letting percent water and soil/silt/clay analysis sit collected

   data and took pictures.

Soil Washing:

1. Filled 15mL tube with 2mL of soil then with 10mL of LB broth.
2. Shook for 15 minutes then spun down at 3000g for 5 minutes.
3. Filtered supernatant with a 22nm filter then added 0.5mL of arthrobacter to

   filtered supernatant.

4. Labeled “Enriched” and placed in shaking incubator for 48 hours.

Data and Observations:
The tree from which I collected soil from was an extremely young sapling. The height was 0.56m, the circumference of the trunk was 3cm, and the canopy diameter was 26cm. There were some damaged browning leaves, but other than that the tree seemed decently healthy.

Mass of soil with water is 4.05g. Mass of sample without water is 3.42g. Mass of water lost is 0.63g divided by the total mass of 4.05g is 15.56% water. The pH of the soil was about 6.5. The soil was 70% silt 30% clay. The amount of sand was too small to measure quantitatively.

Interpretations and Next Steps: Unlike the dead tree I collected from last time this soil is slightly more acidic. Also, there is almost no sand in this soil. There is twice as much moisture in this soil as compared to the previous tree. This could be a confounding variable in the presence of phage in these two samples. I would have to test more trees to see if the lack or presence of phage was due to any of these factors or not. The next step is to conduct a plaque assay to see if this soil has phage in it.